The tumor stromal microenvironment holds exciting potential as a therapeutic target. The major component of solid tumor stroma is the carcinoma associated fibroblast (CAF), which generates structural matrix, stimulates growth, supports neovascularization and promotes metastasis of tumor. Studies suggest various sources for CAFs including tissue fibroblasts, epithelial-to-mesenchymal transdifferentiation (EMT) and bone marrow (i.e., mesenchymal stem cells). We have developed a novel transplantation model in which the bone marrow of lethally irradiated recipient mice is reconstituted by a clonal population of cells derived from a single EGFP+ hematopoietic stem cell (HSC). Our studies using this model demonstrate that CAFs and circulating fibroblast precursors (CFPs) are of HSC origin and promote tumor growth. This novel HSC source for both fibroblast precursors and CAFs is the basis for the proposed studies. It is our hypothesis that HSC-derived CFPs and CAFs play a critical role in tumor proliferation, invasion, migration and metastasis; however the specific role of these HSC-derived CAFs and CFPs in tumor progression has not yet been investigated. Nor has direct comparison of HSC-derived CAFs to non-HSC-derived resident populations been conducted. The proposed studies will profile this unique population of cells and determine their influence on tumor progression using our clonal cell transplantation model in conjunction with Lewis lung carcinoma (LLC) tumor models through the following Specific Aims: 1) To determine the mechanisms by which HSC-derived CFPs and CAFs promote tumor progression. The first goal of this Aim is to compare HSC-derived EGFP+ CAFs and EGFP- resident CAFs via flow cytometric, immunohistochemical and molecular profiling. The effects of HSC-derived CFPs on tumor proliferation, migration and invasion will then be examined in vitro and the tumor-promoting factors responsible identified and validated in gain of function and/or loss of function studies. 2) To identify factors which regulate HSC-derived CFP recruitment, homing, differentiation and maturation in the tumor microenvironment. Studies will examine factors regulating the contribution of HSC-derived CFPs to tumor via functional in vitro experiments. 3) To determine the contributions of HSC-derived CFPs and CAFs to tumor in vivo. These studies will examine the ability of HSC-derived CFPs to enhance LLC tumorigenicity, progression and metastasis. The functional importance of proteins/genes identified in Aims 1 and 2 will also be evaluated using models of genetic deficiency or siRNA methods. Together these studies will not only lead to a better understanding of basic tumor biology, but have the potential to lead to the identification of unique cancer associated molecular signatures and HSC-derived CFP/CAF-specific factors that may be targeted to inhibit solid tumor growth and progression. PUBLIC HEALTH RELEVANCE: Our goal is to determine how hematopoietic stem cell (HSC) carcinoma associated fibroblasts (CAFs) and their circulating fibroblast precursors (CFPs) directly influence tumor progression and identify mechanisms that regulate the contribution of these cells to tumor. Findings from these studies will lead to a better understanding of basic tumor biology as well as identify novel potential therapeutic targets based on HSC-derived CFP/CAF participation in tumor microenvironment. Furthermore, these studies have the potential to lead to the identification of unique cancer associated molecular signatures that may prove useful for stratifying patients and predicting metastasis and clinical outcomes, eventually leading to improved diagnostic and prognostic values.